Over 300 million people in the world suffer from asthma. The US is among the countries with the highest incidence (10.9% of the population) with a yearly cost of $56 billion. Despite these staggering statistics, there have been no new classes of medications targeting airway smooth muscle (ASM)-mediated bronchoconstriction in asthma for many decades. Moreover, long-acting -agonists, the current leading therapy that directly targets ASM constriction, have been associated with an increased mortality from asthma and are currently under FDA-mandated safety review in 5 clinical studies. Our preliminary studies support the hypothesis that drugs targeting the channels and transporters that control chloride flux in ASM may be novel therapies for relaxing ASM. We propose functional and mechanistic studies to document the efficacy of targeting calcium-activated chloride channels alone or in combination with sodium-potassium-chloride co-transporters as novel asthma therapies. We propose that inhaled therapy will circumvent concerns related to systemic toxicity and will confirm existing literature that blockade of these channels also reduces airway mucus production. Thus, we propose the following 3 specific aims: Aim 1a: functional relaxation of ex vivo human and guinea pig ASM by targeting chloride flux pathways. We will demonstrate the effectiveness of blockade of chloride channels alone or in combination with chloride transporters in the prevention of contraction or the induction of relaxation. We will also determine the ability of chloride channel/transporter blockade to potentiate relaxation of 2- agonists in human ASM providing initial translational data for clinically relevant drug discovery. Aim 2: functional reduction of in vivo lung resistance. We will demonstrate that acute aerosol delivery of blockers of chloride channels +/- chloride transporters prevents bronchoconstriction in vivo in 3 mouse models: (1) naively hyperresponsive A/J strain (2) house dust mite sensitized C57 mice, and (3) a mouse with a selective genetic deletion of the TMEM16A chloride channel in smooth muscle. The effect on epithelial mucous production will be assessed in these models. Aim 3: the cellular mechanism(s) by which chloride channels regulate ASM tone. We will demonstrate the link between chloride control of plasma membrane (PM) potential and intracellular signaling pathways linked to contraction/relaxation including stored operated Ca2+ entry, Gq-coupling to inositol phosphate generation, and phosphorylation of contractile-regulatory proteins (RhoA, MYPT1, MLC). We will distinguish between chloride control of Ca2+ flux across the PM vs SR using a FRET-based SR-specific Ca2+ indicator (D1ER).